ES2995063T3 - Bed bugs detection device - Google Patents

Abstract

Embodiments of systems and methods for analyzing a test fluid to detect previous or current bed bug infestations are described. In one embodiment, the method may include receiving the test fluid on a test strip within the detection device. The test strip may include a reaction portion and a reagent portion containing an antibody or antigen-binding fragment that is conjugated to a colored particle. The test fluid may include a bed bug antigen that reacts with the conjugated antibody. The detection device may include first and second optical sensors for monitoring a reaction and a background color intensity, respectively. After a predetermined time delay elapses, the detection device determines whether the bed bug antigen is present in the test fluid using the monitored color intensities and minimum and maximum color intensity thresholds associated with bed bugs. The detection device then generates a result using a visual display. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Bed bug detection device

Field

Embodiments relate generally to detecting a presence of pests, and more particularly, to an electronic detection device, test strip, respective kit and method for detecting household insect pests including, for example, bed bugs.

Background

Studies and surveys over the past decade suggest that bed bug infestations are becoming more prevalent both in the United States and around the world. To begin treating bed bug infestations, pest control operators need effective and accurate procedures to detect and identify the presence of bed bugs.

In a typical example, a pest control operator is called to inspect a housing unit (e.g., a house, apartment, hotel, or hospital) where bed bugs may be present. Current detection options include visual inspection of a suspect room, which is ineffective and unreliable, or obtaining a sample of suspect bed bug residue for analysis at an off-site testing laboratory. Often, the pest control operator may have to use expensive DNA testing equipment off-site to analyze suspect residue for evidence of bed bugs. In addition, bed bug analysis equipment (e.g., DNA analysis equipment) often cannot be used on-site and may require 24 to 48 hours to produce an analysis result. US 8,375,626 describes procedures and reagent strips for detecting bed bugs by using an IgE antibody and colorimetric indicators.

Summary

The present invention relates to an electronic detection device for analyzing a test fluid to determine past or present bed bug infestations, comprising a lateral flow assay test processed within the detection device, wherein the lateral flow assay test comprises a reagent band for receiving the test fluid near a first end of the reagent band, wherein the reagent band comprises: (a) a reagent portion comprising monoclonal antibodies or antigen-binding fragments thereof that are conjugated to colored particles and are capable of binding to bed bug antigens within the test fluid to form bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-122644. [BB2] or Accession Number PTA-122645 [BB7], and (b) a reaction portion comprising immobilized monoclonal antibodies or antigen-binding fragments thereof that are capable of binding to bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7].

The invention further relates to the method of analyzing a test fluid to determine past or present bed bug infestations using the device and kit comprising the same as detailed in the appended claims. In a further aspect, the invention relates to a reagent strip for detecting past or present bed bug infestations comprising, in order, a first end, a reagent portion containing colored particles, a reaction portion, and a second end, wherein the reagent strip is configured to receive a test fluid near the first end and: (a) the reagent portion comprises monoclonal antibodies or antigen-binding fragments thereof that are conjugated to the colored particles and are capable of binding to bed bug antigens within the test fluid to form bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number pTa-122644 [BB2] or Accession Number PTA-122645 [BB7], and (b) the reaction portion comprises monoclonal antibodies. immobilized or antigen-binding fragments thereof that are capable of binding to bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7].

Additional embodiments are summarized in the appended dependent claims.

In accordance with certain disclosed aspects of the present disclosure, a system and method are provided for determining, by means of a detection device, whether the presence of one or more insect pests is detected. The detection device may be configured to store pest profiles, each with thresholds for one or more characteristics of the profile. In one embodiment, the detection device detects, within a certain time, the presence of a sample fluid (or test fluid) being analyzed during a lateral flow assay processed within the detection device. The detection device further checks that a sufficient amount of colored particles has passed through a test zone such that the presence of one or more pests within the sample fluid can be detected. The disclosed device then compares the test results to one or more pest profiles to determine whether one or more pests are present.

In accordance with the invention, a detection device receives a test fluid near a first end of a reagent strip within the detection device, wherein the test fluid flows past a reagent portion of the reagent strip containing colored particles and through a reaction portion of the reagent strip, wherein the reagent portion contains antibodies conjugated to colored particles, and wherein the conjugated antibodies are capable of binding with any bed bug antigens within the test fluid to form bed bug molecules. A first optical sensor within the detection device monitors the reaction color intensity of the reaction portion of the reagent strip when antibodies immobilized within the reaction portion bind to bed bug molecules. A second optical sensor within the detection device monitors a background color intensity of a portion of the reagent strip proximate the reaction portion. The detection device determines that an initial amount of test fluid has passed through the reaction portion based on the monitored reaction color intensity and the monitored background color intensity. The detection device determines that a given amount of colored particles of the reagent portion has passed through the reaction portion based on the monitored reaction color intensity and the monitored background color intensity. The detection device detects that a delay time has elapsed since the given amount of colored particles was determined to have passed through the reaction portion. The detection device determines a bed bug profile result by using the monitored color intensities and the minimum and maximum color intensity thresholds, wherein the time delay, the minimum color intensity threshold, and the maximum color intensity threshold are stored in a memory of the detection device, and wherein the profile result indicates whether the presence of bed bugs has been detected in the test liquid. The detection device then outputs the bed bug profile result by means of a visual display.

In certain embodiments, the antibody is produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-122644 [BB2], or an antigen-binding fragment thereof.

In certain embodiments, the antibody is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], or an antigen-binding fragment thereof.

The antibody used in the electronic device and reagent-reactive band of the invention is a monoclonal antibody or an antigen-binding fragment thereof comprising the heavy chain and light chain complementarity determining regions (CDR) of an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chain variable regions of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the heavy and light chains of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7].

In certain embodiments, the antibody or antigen-binding fragment of any of the preceding embodiments is capable of binding to a bed bug antigen in a whole bed bug lysate or a collection paper extract comprising bed bug litter material.

In certain aspects, the antibody is a mutant of an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or a mutant of an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], wherein the mutant is capable of binding to a bed bug antigen in a whole bed bug lysate or a collection paper extract comprising bed bug litter material.

In certain embodiments, the antibody is a conjugated monoclonal antibody or a conjugated antigen-binding fragment comprising any of the antibodies, antigen-binding fragments, or mutants and a detection agent. In certain embodiments, the detection agent is colloidal gold. In certain embodiments, the conjugated antibody or the conjugated antigen-binding fragment comprises an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], or an antigen-binding fragment thereof, or an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], or an antigen-binding fragment thereof.

Certain aspects may include a composition comprising any of the antibodies, antigen-binding fragments, mutants, or conjugated antibodies or conjugated antigen-binding fragments, or a combination thereof. In certain aspects, the composition comprises an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the composition comprises an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], and a conjugated antibody comprising the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7] and a detection agent. In certain aspects, the composition comprises an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], and an antibody conjugate comprising the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and a detection agent.

Certain embodiments may include a kit comprising any of the above inventions or embodiments, or a combination thereof.

Certain aspects may include a hybridoma capable of producing an antibody, wherein the hybridoma is deposited with the ATCC under Accession Number PTA-122644 [BB2] or wherein the hybridoma is deposited with the ATCC under Accession Number PTA-122645 [BB7].

Certain aspects may include an isolated cell that produces an antibody, an antigen-binding fragment, or a mutant.

Certain aspects may include a method of manufacturing an antibody, antigen-binding fragment, or mutant, comprising culturing an isolated cell that produces the antibody, antigen-binding fragment, or mutant, and isolating the antibody, antigen-binding fragment, or mutant from the cultured cell.

Certain aspects may include a method of detecting bed bugs, comprising contacting a sample comprising a bed bug antigen with any of the antibodies, antigen-binding fragments, mutants, conjugated antibodies or conjugated antigen-binding fragments, or compositions or a combination thereof, and detecting the binding of the bed bug antigen to the antibody or antigen-binding fragment, mutant, conjugated antibody or conjugated antigen-binding fragment, composition or combination thereof. In certain aspects, the sample is contacted with an antibody of any of the above inventions or embodiments and a conjugated antibody. In certain embodiments, the antibody is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], and the conjugated antibody comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7] and a detection agent. In certain embodiments, the antibody is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], and the conjugated antibody comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and a detection agent. In accordance with the invention, detecting comprises performing a lateral flow assay. In certain aspects, the method further comprises collecting a sample comprising the bed bug antigen with a collection device and extracting antigen from the sample. In certain embodiments, the collection device is a swab.

Brief description of the drawings

FIG. 1 is a diagram of a detection device for detecting the presence of bed bugs.

FIG. 2 is an exploded view of the detection device of FIG. 1.

FIG. 3 is an enlarged elevation view of the sensing device of FIG. 1.

FIG. 4 is an enlarged sectional view of the sensing device of FIG. 3 before a portion of the cap is removed.

FIG. 5 is a fragmentary sectional elevation view of the device of FIG. 3 when the cap portion is removed.

FIG. 6 is an enlarged sectional view of FIG. 5 after a portion of the cap has been removed.

FIG. 7 is a block diagram illustrating the components of a sensing device processor.

FIGS. 8 to 13 are flow charts illustrating procedures performed by a detection device.

FIG. 14 is a diagram illustrating examples of results obtained in lateral flow assays of samples analyzed by a detection device.

FIG. 15 shows the results of a sandwich capture assay using the anti-bed bug monoclonal antibodies BB2 and BB7. Nitrocellulose bands are shown with bed bug antigen binding indicated by dots associated with the presence of the gold-conjugated BB2 or BB7 monoclonal antibody (“Detector Ab”), with the pH of the gold-conjugated antibody as indicated. Reactive bands are indicated with “bed bug” for the antigen. Negative control bands are indicated with “none” for the antigen, with PBS added to the bands in place of the bed bug antigen. Capture antibodies associated with lanes 1 through 3 were the rabbit polyclonal antibody against bed bug (lane 1), the BB2 monoclonal antibody (lane 2), and the BB7 monoclonal antibody (lane 3).

FIGS. 16-21 show the results of a lateral flow immunoassay for swab samples obtained from the noted bed bug infestation levels, with level 0 containing no bed bugs and level 8 containing the highest level of bed bugs. Swab samples were extracted in Extraction Buffer 1 containing 1X Tris-HCl (pH 7.6), 0.05% NaN<3>, 0.1% BSA, and 0.1% Tween-20, and the noted dilutions were applied to the reactive bands. “Buffer” indicates a negative control reactive band to which Buffer 1 was applied in place of a swab sample. All bands contain a positive control band of goat anti-mouse antibody, which is located above a band of BB7 capture antibody for bed bug antigen binding. The lanes become visually detectable only upon binding of the gold-conjugated BB2 mouse monoclonal antibody to the goat anti-mouse positive control lane or to the immobilized bed bug antigen captured by the BB7 lane. The “Buffer” band line and the corresponding lines of the reactive bands are the positive controls indicating binding of the goat anti-mouse antibody to the gold-conjugated BB2 detector antibody. Only the positive controls are detectable in FIG. 16 due to the absence of antigen. The lines below the positive control lines in FIGS. 17 through 21 indicate binding of the gold-conjugated BB2 antibody to the immobilized bed bug antigen captured by the BB7 lane. FIGS. 20 and 21 show noticeable dirt and debris for the less diluted samples at the bottom of the reactive bands associated with the highest levels of bed bug infestation.

FIGS. 22A and 22B are graphs based on measurements made by the Axxin reagent strip reader of swab samples for levels 2, 4, and 8 extracted in buffer 1. In FIG. 22A, the “concentration” x-axis is the dilution associated with the measured swab samples, and the y-axis is the “test line area” value provided by the Axxin reagent strip reader. In FIG. 22B, the value obtained for buffer 1 as a negative control (i.e., “Bo”) was divided by itself to obtain a normalized value of 1. The negative control (Bo) reading was then divided by the area of the test lines (B) for each dilution of the test sample in the level, with smaller values below 1 suggesting higher amounts of bed bug antigen and values above 1 indicating no bed bug antigen.

FIGS. 23-25 show the results of a lateral flow immunoassay for swab samples obtained from the noted bed bug infestation levels. The swab samples were extracted in extraction buffer 2 containing 1X Tris-HCl (pH 7.6), 0.05% NaN<3>, 0.1% BSA, and 0.2% Tween-20, and the noted dilutions were applied to the reagent bands. FIG. 25 shows noticeable dirt and residue for the less dilute samples at the bottom of the reagent bands associated with the highest bed bug infestation levels. Labeling of the bands and interpretation of the results are as described for FIGS. 16-21.

FIGS. 26A and 26B are graphs based on measurements made by the Axxin reagent strip reader of swab samples for levels 4, 5, and 8 extracted in buffer 2. The labeling of the graphs is as described for FIGS. 22A and 22B.

FIGS. 27-31 show the results of a lateral flow immunoassay for swab samples obtained from the noted bed bug infestation levels. The swab samples were extracted in extraction buffer 3 containing 1X Tris-HCl (pH 7.6), 0.05% NaN3, 0.25% BSA, and 0.1% Tween-20, and the noted dilutions were applied to the reagent bands. FIGS. 30 and 31 show noticeable dirt and residue for the less dilute samples at the bottom of the reagent bands associated with the highest bed bug infestation levels. Labeling of the bands and interpretation of the results are as described for FIGS. 16-21.

FIGS. 32A and 32B are graphs based on measurements made by the Axxin reagent strip reader of swab samples for levels 2, 5, and 8 extracted in buffer 3. The labeling of the graphs is as described for FIGS. 22A and 22B.

FIGS. 33A and 33B are graphs based on measurements made by the Axxin reagent strip reader showing a comparison of results from level 8 swab samples extracted in buffers 1, 2, and 3. The labeling of the graphs is as described for FIGS. 22A and 22B.

FIG. 34 is a graph based on measurements made by the Axxin reagent strip reader for nine replicates of Extraction Buffer 1 as a negative control and nine replicates each of 1/2048, 1/512, and 1/128 dilutions for the Level 7 swab samples extracted in Buffer 1. The x-axis indicates the nine replicates by “run” number; the y-axis is as described for FIG. 22A.

The drawing in which an element first appears is typically indicated by the leftmost digit or digits in the corresponding reference numeral. In the drawings, similar reference numerals may indicate identical or functionally similar elements.

Detailed description

What is needed is a portable and reliable detection device that can be used on-site to identify and detect the presence of bed bugs within minutes. In accordance with certain disclosed aspects of the present disclosure, a system and method are provided for determining, by means of a detection device, whether the presence of one or more insect pests is detected. The detection device may be configured to store pest profiles, each with thresholds for one or more characteristics of the pest profile. In one embodiment, the detection device detects, within a certain time, the presence of a sample fluid being analyzed during a lateral flow assay processed within the detection device. The detection device further verifies that a sufficient amount of colored particles has passed through a test zone such that the presence of one or more pests within the sample fluid can be detected. The disclosed device then compares the test results to one or more pest profiles to determine whether one or more pests are present.

Implementation of the system

FIG. 1 is a diagram illustrating a detection device 100 for detecting the presence of bed bugs. Detection device 100 may be implemented similarly to a testing device described in U.S. Patent No. 7,220,597 entitled “Assay Test Device and Method of Making Same", and U.S. Patent No. 7,214,542 entitled “Method of Processing Assay Test Results".

As shown, the detection device 100 may include an elongated housing 12 adapted to be held in the hand of a user, such as a pest control operator. In one embodiment, the housing 12 may include a removable cap 14 further attached to the switch actuator 47 extending through the opening 52. As detailed in FIG. 5 below, when the removable cap 14 is removed, the detection device 100 may be activated (e.g., turned on) to begin running a detection software program to identify and detect the presence of bed bugs. In one embodiment, in place of the removable cap 14, the detection device 100 may include the opening 13 such that the detection device 100 may be activated when light entering the opening 13 is detected. In one embodiment, the detection device 100 may include a switch or button to activate the detection device 100.

The result display 25 may include a yellow light emitting diode (LED) 27, a green LED 29, and a red LED 32 arranged in a row on the top surface of the housing 12 and positioned within corresponding openings 34, 36, and 38 of FIG. 2. One or more of the LEDs 27, 29, and 32 may be controlled by the processor 21 of FIG. 2 to indicate, for example, by lighting, whether a detection result indicates the presence of bed bugs or whether the detection result is indeterminate. In one aspect, an LED display, LCD display, or other display electronics may be used to display the detection result.

FIG. 2 is a diagram illustrating an exploded view of the sensing device 100 of FIG. 1. FIG. 3 further illustrates an enlarged elevational view of the sensing device 100 of FIG. 1. The housing 12 may include the lower portion 69 that is attached to the upper portion 72 to enclose the printed circuit board 19 and the elongated reactive strip 16 longitudinally disposed within the housing 12. Also shown is the removable cap 14 connected to the switch actuator 47, which includes the insulating strip 49, which may be in the form of a rigid strip of suitable materials, such as thermal plastic or other similar material. The sensing device 100 may be activated when the insulating strip 49 is removed from and through the opening 52.

The reagent strip 16 may be in the form of the reagent strip disclosed in European Patent Application No. EP0,962,771A1. In one embodiment, the reagent strip 16 provides an environment for performing a lateral flow assay or immunoassay test used by the detection device 100 to determine whether bed bugs are present or detected. Depending on the type of reagent strip 16 and the materials/components incorporated on or within the reagent strip 16, the detection device 100 may also detect the presence of other types of insect pests (e.g., cockroaches or termites) or whether multiple pests, including bed bugs, are present.

To perform a lateral flow assay, the reagent strip 16 includes a backing strip 54 having a sample pad or wick 56 for receiving and absorbing a sample fluid potentially containing bed bug antigens. For example, the sample fluid may be an extract produced from a sample potentially comprising bed bug residues, such as litter and/or fabrics. In one embodiment, to obtain the sample fluid, a pest control operator may first use a swab material (e.g., a cotton swab) to swab bed bug hot spots at a given location. Examples of bed bug hot spots may include bed sheets, mattress frames, mattress legs, electrical outlets, window sills, furniture surfaces, or other regions proximate to a sleeping area (e.g., a mattress, futon, or couch). After cleaning bed bug foci, the pest control operator may dip the cleaning material, which may contain bed bug residue, into an extraction buffer (i.e., a liquid that can extract bed bug antigens from a sample) within a jar or other container to produce a sample fluid. The pest control operator may then drip portions of the buffer liquid onto a sample pad or wick 56, which receives the buffer liquid and wick mixture as the sample fluid.

In one aspect, where the sensing device 100 includes a removable cap 14, the reagent strip 16 may extend outside of the housing 12 and be covered by the removable cap 14 or a lid portion when assembled to the housing 12. When the removable cap 14 is removed from the housing 12, the wick 56 is exposed for application of sample fluid as described. In an embodiment without a removable cap 14, the wick 56 may instead receive sample fluid applied through the opening 13 as depicted in FIG. 1.

The reagent strip 16 includes a porous carrier strip 58 having a reagent section or pad 61 attached to the wick 56, and a fluid absorption section or absorption pad 63 at the opposite end portion of the reagent strip 16 from the wick 56. The porous carrier strip 58 allows sample fluid from the wick 56 to flow through the reagent pad 61 and the line forming zone 65 to the absorption pad 63, where excess sample fluid is absorbed.

A capture section or line forming zone 65 on the upper surface of an intermediate portion of the porous carrier web 58 is disposed opposite a reaction sensor or front sensor 23. In the line forming zone 65, a line of specific intensity and coloration is formed once a reaction with bed bug antigens occurs if there is a sufficient amount of bed bug antigen in the sample fluid to indicate that bed bugs are present within a room suspected of harboring bed bugs. In one aspect, the detection device 100 may read a measurement from the front sensor 23 to determine whether the presence of bed bugs is detected. In one embodiment, to increase accuracy and reduce (or eliminate) calibration time, the detection device 100 may utilize measurements from more than one sensor, such as the front sensor 23 and the rear sensor 18.

In one embodiment, the reagent pad 61 may contain a solubilizable mixture of bed bug antibodies conjugated to colored particles. Examples of colored particles may include, for example, colloidal gold, latex microspheres, or fluorescent labels. A colored particle is used as a detection agent such that the more colored particles the detection device 100 detects during the profile evaluation (to be described below with respect to FIG. 7 ), the greater the likelihood that a pest, such as bed bugs, is present in the sample. As the sample fluid received on the wick 56 migrates through the reagent pad 61 to an intermediate portion of the support band 58, the sample fluid may mix with the solubilizable mixture on the reagent pad 61 to form a sample-conjugate mixture. Bed bug antigens present in the sample fluid may bind to the conjugated anti-bed bug antibodies to form sample-conjugate molecules. As the sample fluid dissipates or solubilises the solubilizable mixture, the sample-conjugate fluid flows toward the absorption pad 63 until a background sensor or back sensor 18 disposed opposite the intermediate portion of the carrier web 58 detects the presence of the sample-conjugate fluid due to the color change of the wetted porous carrier web 58. In one aspect, to increase accuracy and reduce (or eliminate) calibration, the sensing device 100 determines a relationship between measurements from more than one sensor, including the front sensor 23 and the back sensor 18, to determine whether the sample-conjugate fluid is present.

To enable measurements to be taken from the sensors, a light emitting diode (LED) 67 may be disposed or mounted on the underside of the printed circuit board 19 between the rear sensor 18 and the front sensor 23 to illuminate the intermediate portion of the porous carrier strip 58 and reflect light therefrom toward the sensors. The illumination LED 67 may produce light in the visible range of the electromagnetic spectrum. A white LED may provide more accurate measurements, but another color LED, such as a green LED, may also be used as the illumination LED 67, depending on the color of the line formed in the line forming region 65. In one aspect, the sensing device 100 may cause the LED 67 to turn on whenever a measurement is to be read from the rear sensor 18 or the front sensor 23.

The line-forming zone 65 may include a region (e.g., a line) of one or more types of immobilized antibodies that can bind to the bed bug antigens of the sample conjugate molecules within the sample conjugate fluid flowing past the line-forming zone 65. In one embodiment, the immobilized antibodies may include the portion of antibacterial antibodies conjugated on or within the reagent pad 61. As the sample-conjugate fluid from the reagent pad 61 flows through the line-forming zone 65 toward the absorption pad 63, an increased amount of the sample-conjugate molecules may react (or bind) to the immobilized antibodies. In one embodiment where the conjugate particle is a colored particle such as colloidal gold, as increased reaction occurs within the line forming zone 65 due to increased presence of bed bug antigens within the sample fluid, the coloration within the line forming zone 65 may be intensified at a greater rate. In one embodiment, depending on the type of conjugate particle, a level of luminescence or fluorescence may be intensified instead.

The rear sensor 18 (background sensor) and the front sensor 23 (reaction sensor) may be mounted on the underside of the printed circuit board 19. The front sensor 23 may be disposed directly in front of the line-forming region 65 of the reagent strip 16 to pick up a colorimetric reflectance of the reaction, i.e., a color intensity of the line-forming region 65. The rear sensor 18 may be disposed near the line-forming region 16 (but not directly on the line-forming region 16) between the line-forming region 16 and the absorption pad 63. In one embodiment, the rear sensor 18 may be disposed between the line-forming region 16 and the reagent pad 61. The rear sensor 18 similarly measures or picks up a color intensity of the region of the reagent strip disposed in front of the rear sensor 18. Although the sensing device 100 may use only the readings from the rear sensor 18 to detect the color intensity of the reaction, the front sensor 23 may be disposed in front of the line-forming region 65 of the reagent strip 16. the presence of the conjugate fluid as it flows along the reagent band 16, in an improved embodiment, the detection device may utilize the measurement readings from both the rear sensor 18 and the front sensor 23. Similarly, the detection device 100 may utilize the readings from the front sensor 23 or from both the rear sensor 18 and the front sensor 23 to determine whether the presence of bed bugs is detected in the line-forming zone 65.

Each of the rear 18 and front 23 sensors may be a photo-optic sensor including, for example, a photoconductive cell or a light-dependent resistor that varies resistance based on a detected and measured incident light intensity due to colorimetric diffuse reflectance within the line-forming region 65. A measured colorimetric diffuse reflectance may be representative of the color intensity within the line-forming region 65. For example, as the color within the line-forming region 65 intensifies, more light may be absorbed by the line-forming region 65, i.e., less light may be reflected by the line-forming region 65, and a photoconductive cell may emit a higher resistance due to a lower incident light intensity. In one embodiment, the photo-optic sensor may include a photodiode that produces a leakage current that is directly proportional to the incident light intensity. Depending on the conjugated particle, other types of sensors may be used, such as a Hall effect sensor to capture the magnetic flux density.

The power supply generally indicated in power region 41 is mounted on the upper surface of printed circuit board 19. Power region 41 may include battery 43 which electrically powers printed circuit board 19 and its components, including, for example, yellow LED 27, green LED 29, red LED 32, rear sensor 18, illumination LED 65, front sensor 23, and processor 21.

The processor 21 may be mounted on the top surface of the printed circuit board 19 and programmed to begin detecting the presence of bed bugs when the detection device 100 is activated or turned on. The processor 21 may include input ports for receiving power and reading measurements from the front sensor 23 and the rear sensor 18 and include output ports for controlling the illumination LED 65 and the LEDs 32, 27, and 29 of the results display 25. In one embodiment, the processor 21 may be a microprocessor (e.g., an 8- or 16-bit microcontroller) with limited processing and memory capabilities due to the size, power, and cost limitations of the detection device 100. To efficiently detect the presence of bed bugs given the limited hardware, the processor 21 may include stored algorithms for efficient calibration processing and accurate bed bug detection results. In one aspect, the processor 21 may detect different types of insect pests or the presence of more than one type of insect pest, including bed bugs.

FIGS. 4 through 6 are diagrams illustrating configurations within the sensing device 100. As shown in FIG. 4 is an enlarged sectional view of the sensing device of FIG. 3 prior to removing the removable cap 14 and powering the sensing device 100. FIG. 5 illustrates a fragmentary sectional elevational view of the device of FIG. 3 when the removable cap 14 is removed. FIG. 6 illustrates an enlarged sectional view of FIG. 5 after the removable cap 14 has been removed.

In one aspect, the switch actuator 47 in the form of an insulating strip 49 extends through an opening 52 in an angle wall portion of the housing 12 to the switch 45 when the removable cap 14 is assembled to the housing 12 as shown in FIGS. 1 and 3. When the removable cap 14 is removed from the housing 12 as indicated in FIG. 5, the switch actuator 47 may be removed from the switch 45 as indicated in FIGS. 4 and 6 to cause the battery 43 to be electrically connected to the printed circuit board 19 to energize or turn on the sensing device 100.

Although the detection device 100, as shown, may be a single-use device that is accurate and rapid in its determination of the presence of bed bugs, the detection device 100 may be employed as a multiple-use device. For example, a multiple-use detection device 100 may include a housing 12 that may be removed as indicated in FIG. 2 to allow the reagent strip 16 to be replaced with a new reagent strip to perform additional lateral flow testing. The insulating strip 49 of the switch actuator 47 may be in the form of a rigid strip of suitable materials, such as thermal plastic or other similar material. In this regard, the insulating strip 49 may be reinserted through the opening 52 and under the battery 43 to electrically decouple the battery 43 from the printed circuit board 19.

FIG. 7 is a block diagram 700 illustrating the components of the processor 700 of a sensing device, such as the sensing device 100 of FIGS. 1-3. The processor 700 may be an exemplary implementation of the processor 21 of FIGS. 2, 3, and 5. The processor 702 may include a memory 704, ports 714A and B, a clock 728, and components 730-742.

Ports 714A may be input ports, including power port 720, which powers processor 702 from a power source, such as battery 43 of printed circuit board 19 of FIG. 2. Input ports 714A may further include front sensor port 716 and rear sensor port 718, which receive measurements from front sensor 23 and rear sensor 18 of FIG. 2, respectively. Ports 714B may include output ports for outputting a result indicating whether processor 702 encountered an error and whether processor 702 determined the presence of one or more pests including, for example, bed bugs. As shown, output ports 714B may include yellow LED port 722, green LED port 724, and red LED port 726 for controlling yellow LED 27, green LED 29, and red LED 32 of FIG.

2, respectively. In one embodiment, a single LED port may drive more than one LED.

The memory 704 may include registers, RAM, ROM, cache, or other types of memory storage. The memory 704 stores the pest profile 706, the operating parameters 712, the counter 709, and the readings from the sensors 713A and B. The operating parameters 712 may include a liquid threshold, a color threshold, and minimum and maximum thresholds for each of the front 23 and rear 18 sensors. The liquid threshold may be representative of a minimum initial amount or concentration of sample-conjugate fluid flowing through the line formation zone 65 that may be required before the processor 702 continues to determine whether bed bugs are detected. The color threshold may be representative of a color intensity value proportional to a minimum amount or concentration of sample-conjugate fluid flowing through the line formation zone 65 that may be necessary to validate any final detection determination made by the processor 702. Other operating parameters 712 may include a range of power values (e.g., minimum and maximum power voltages) such that any power value outside the range indicates that the processor 702 is not being adequately powered through the power port 720, and a range of sensor values (e.g., minimum and maximum sensor values) such that sensor values outside the sensor range are invalid. Depending on the type of sensor used or the measurement methodology, the sensor range values may be, for example, resistances, currents, or voltages. For example, a sensor can be a photodiode (i.e. a device that converts received light into current) or a photoconductive cell (i.e. a device that converts received light into resistance). The measured sensor value for each sensor type can be, for example, a voltage value.

Counter 709 may be a register that is incremented by clock 728 of processor 702. A counter value of counter 709 may be representative of a period of time or clock cycles since counter 709 was activated or initialized. In one embodiment, the counter value may be representative of a period of time since counter 709 was cleared or set to 0 by processor 702.

The pest profile 706 may include thresholds or parameters configured for profile characteristics of a specific insect pest, such as bed bugs, associated with the pest profile 706. The profile characteristics may include a test time period and a measured color intensity range. As shown, the configured thresholds include intensity threshold 708 and test time threshold 710, such that the processor 702 determines the presence of a pest when the processor 702 calculates a result value that exceeds intensity threshold 708 after a value of counter 709 exceeds test time threshold 710. In one embodiment, intensity threshold 708 may include a range of intensity thresholds, such as a minimum threshold and a maximum threshold, such that the processor 702 indicates a detected presence of the pest only when a calculated result value exceeds or reaches the maximum threshold. If the calculated value falls below the minimum threshold, the processor 702 may indicate the absence of the pest. However, if the calculated value falls between the minimum and maximum thresholds, the processor 702 may indicate or output an indeterminate result to reduce the number of false positives, i.e., incorrectly determining the presence of the pest. Similarly, the test time threshold 710 may include a range of test time thresholds, such as a minimum test time threshold and a maximum test time threshold. A test result determined by the processor 702 can only be accurate if measurements between the minimum and maximum test time thresholds are used.

Pest profile 706 may include two or more pest profiles for respective insect pests. Each pest profile may include, for at least one profile characteristic, a threshold range that does not overlap or intersect with a corresponding threshold range (i.e., threshold range of the same profile characteristics) of any other pest profile. For example, pest profile 706 may include a bed bug profile and a cockroach profile. The bed bug profile may be configured to include, for example, test time thresholds 710 of 3 min and 5 min and intensity thresholds 708 ranging from 18 to 100. In contrast, the cockroach profile may include intensity thresholds 708 of 15 to 30 that overlap the bed bug profile's intensity thresholds of 18 to 30. However, the cockroach profile may also include test time thresholds 710 of 9 min to 10 min that do not overlap any of the test time thresholds 710 of all other pest profiles 706, including the bed bug profile (e.g., 9 to 10 min does not overlap 3 to 5 min in the bed bug profile).

The sensor readings 713A and 713B may store the readings of the front sensor 23 and the rear sensor 18, respectively. The sensor readings 713A and the sensor readings 713B may each include respective initial sensor readings and respective subsequent readings of the front sensor 23 and the rear sensor 18, respectively. An initial reading of the front sensor 23 and the rear sensor 18 may be updated depending on when the processor 702 executes the color checking component 736 (to be described later). By tracking subsequent sensor readings in association with a tracking time throughout the pest detection process, the processor 702 may determine and detect whether two or more pest profiles 706 have been met, and indicate or output the respective results of the determination. In one embodiment, a pest profile 706 is met or satisfied if each configured threshold within that same pest profile 706 is met.

The processor 702 may include various components 730-742 for implementing the sensing functionality of the sensing device 100. For ease of understanding, the component descriptions of FIG. 7 will refer to FIGS. 1-3, which illustrate examples of physical structures or components of the sensing device 100. A component of the processor 702 may include a selection of stored operations that, when executed on the processor 702, cause the processor 702 to perform the operations of the component.

FIG. 8 is a flow diagram of a method 800 for a generalized algorithm for detecting whether one or more insect pests are present. The method 800 may be carried out by processing logic within one or more components, such as components 730-742 of processor 702 of FIG. 7, which may comprise hardware (e.g., circuitry, dedicated logic, programmable logic, microcode, etc.), software (e.g., instructions executing on a processing device), or a combination thereof. For ease of reference, descriptions of the following steps may refer to components of FIG. 7 and structures within FIG. 2.

In step 802, the power-on test component 732 activates the detection device 100. Once activated, the detection device 100 begins analyzing a lateral flow assay for the presence of one or more insect pests, including bed bugs. The power-on test component 732 may activate the detection device 100 when, for example, the detection device 100 is turned on or powered. The power-on test component 732 may additionally launch a self-test sequence to verify an operating state of the detection device 100, described further with respect to FIG. 7 below. In one embodiment, the power-on test component 732 turns on or flashes one or more LEDs to indicate that step 802 is being performed. For example, the power-on test component 732 may flash the green LED 29 and flash the yellow LED 27.

In step 804, upon completion of step 802, the power-on test component 732 takes (or requests the sampling component 740 to take) an initial front sensor reading and an initial rear sensor reading. These initial readings may be stored in sensor readings 713A and B.

In step 806, the liquid monitoring component 734 monitors an initial amount of detected liquid flowing past the line formation zone 65. The liquid may be a conjugated sample fluid flowing from the reagent pad 61 into the absorption pad 63, described with respect to FIG. 2 above. Since peak detection and profile evaluation (as explained with respect to FIG. 7 below) may be time critical, the detection device 100 should only begin tracking an elapsed time when it is detected that a lateral flow assay test is being performed. For example, based on the specific conjugated anti-bed bug antibody and the concentration thereof in the reagent pad 61, a determination of the presence of bed bugs made by the processor 700 may only be valid within a certain elapsed time interval from when the lateral flow assay test was initiated.

In step 808, the color checking component 736 checks whether sufficient color particles within a sample-conjugate fluid have passed the line forming zone 65. When colloidal gold is used, this may be referred to as checking for “gold migration.” In one embodiment, if very little of the sample-conjugate fluid has migrated beyond the line forming zone 65, then any subsequent profile evaluation results may be invalid. This is because the processor 702 determines a bed bug presence, i.e., a test result satisfies the bed bug profile, based on at least one detected color intensity of the line-forming zone 65. Thus, in one example, although bed bug residue may be present in the sample-conjugate fluid, a color intensity of the line-forming zone 65 may not meet a minimum intensity threshold if not enough, for example, colloidal gold has passed through the line-forming zone 65. In one aspect, the color check component 736 may turn on or flash one or more LEDs to indicate that step 808 is being performed. For example, the color check component 736 may flash the red LED 32 and flash the yellow LED 27.

In step 810, the profile evaluation component 742 determines a profile evaluation result for the pest profile 706, such as a bed bug profile. In one aspect, only one profile exists for evaluation. In another aspect, more than one profile may be configured in the pest profile 706, and the profile evaluation component 742 determines a profile evaluation result for each insect pest that has a configured pest profile. The profile evaluation result may be one of three possibilities: presence of insect pest detected, absence of insect pest detected, and indeterminate result.

In one aspect, if any of steps 804 through 810 return an invalid result, method 800 proceeds to step 818. In step 818, processor 702 may control one or more LEDs to indicate that an error has occurred. For example, processor 702 may turn on yellow LED 27. In one aspect, the turned on LED may continue to turn on until battery 43 of sensing device 100 is depleted.

Steps 812 through 816 include possible LED outputs indicating a valid pest profile result from step 810. In one aspect, profile evaluation component 742 may indicate the result of a pest profile through the use of a variety of output transducers, including one or more LEDs, LCDs, speakers, or haptic feedback motors. In step 812, when the result of the profile evaluation of step 810 is negative or the absence of insect pests is detected, the profile evaluation component 742 turns on the green LED 29. In step 814, when the result of the profile evaluation of step 810 is indeterminate, the profile evaluation component 742 turns on the green LED 29 and the red LED 32. And in step 816, when the result of the profile evaluation of step 810 is positive or the presence of an insect pest is detected, the profile evaluation component 742 turns on the red LED 32. The method 800 ends in step 820. FIGS. 9-13 are flowcharts detailing various steps of method 800. Methods 900-1300 may be performed by processing logic within one or more components, such as the components of processor 702 of FIG. 7, which may comprise hardware (e.g., circuitry, dedicated logic, programmable logic, microcode, etc.), software (e.g., instructions executing on a processing device), or a combination thereof. For ease of reference, descriptions of the following steps may refer to components of FIG. 7 and structures within FIG. 2.

FIG. 9 is a flow diagram of a method 900. Method 900 details the operations of step 806, which checks for the presence of liquid, in accordance with an exemplary embodiment. In step 902, liquid check component 734 initializes, resets, or clears counter 709.

In step 904, the fluid check component 734 invokes the sampling component 740 to read measurements from the rear sensor 18 and the front sensor 23. The fluid check component 734 receives either a calculated result (Calc) or an indication that an error has occurred, i.e., that the calculated result is invalid. If an error occurs, the fluid check component 734 returns an invalid result in step 914.

In step 906, the liquid check component 734 determines whether the received calculated result exceeds a liquid threshold within the operating parameters 712. If the liquid threshold has been exceeded, the liquid check component 734 returns a valid result in step 916. A calculated result that exceeds the liquid threshold may indicate that a lateral test is being performed for, for example, bed bug detection.

In step 908, the liquid check component 734 determines whether a maximum time (e.g., 24 hours) has elapsed. If so, the liquid check component 734 returns an invalid result in step 914.

In step 910, the fluid check component 734 determines whether the initial front and rear sensor readings need to be retaken. In one embodiment, the fluid check component 734 determines whether the following inequality holds: “|Front Reading - Initial Front Reading| > DRIFT and |Calc| < MIN.” A current front sensor reading that strays too far from the stored initial front reading, for example, exceeds a DRIFT value of 20, must be recalibrated if the calculated result (Calc), is less than MIN, such as 3. A calculated result below a specified minimum, for example, 3, may indicate that the calculated result is caused by noise in the sensor measurements.

In step 912, the fluid check component 734 recalibrates the front and rear sensors by adjusting the initial front and rear readings to a current front reading and a current rear reading, respectively.

FIG. 10 is a flow chart of a method 1000. The method 1000 details the operation of step 808 of checking for sufficient colored particles such as colloidal gold, in accordance with an exemplary embodiment. In step 1002, the liquid checking component 734 initializes, resets, or clears the counter 709.

In step 1004, the color check component 736 invokes the sampling component 740 to read measurements from the rear sensor 18 and the front sensor 23. The color check component 736 receives either a calculated result (Calc) or an indication that an error has occurred, i.e., that the calculated result is invalid. If an error occurs, the color check component 736 returns an invalid result in step 1018.

In step 1006, the color check component 736 invokes the peak detection component 738. The peak detection component 738 checks for the existence of a peak, and the color check component 736 receives a peak detection result from the peak detection component 738. Peak detection is described below with respect to FIG. 12.

At step 1008, if the result of the peak detection indicates that no peak was found, procedure 1000 proceeds to step 1012. Otherwise, procedure 1000 proceeds to step 1010.

In step 1010, the color check component 736 determines whether the calculated result (Calc) exceeds the COLOR THRESHOLD parameter of the operating parameters 712. The COLOR THRESHOLD may indicate that a sufficient amount or concentration of colored particles has been detected within the sample-conjugate fluid as the lateral flow assay proceeds. When the calculated result exceeds the COLOR THRESHOLD, the color check component 736 returns a valid result in step 1016. Otherwise, the method 1000 proceeds to step 1012 where the counter 709 is incremented. In one embodiment, instead of step 1012, the counter 709 may be incremented by clock 728 while the color check component 736 performs steps 1004 through 1010 and 1014 through 1018.

In step 1014, the color check component 736 checks whether a value of the counter 709 exceeds a MAXIMUM DELAY, for example, 172 seconds or a corresponding number of clock cycles.

FIG. 11 is a flow diagram of a method 1100. The method 1100 details the operation of the sampling component 740 (invoked by one or more steps of the method 800 of FIG. 8) to read sensor values and calculate results. The method 1100 begins at step 1102.

In step 1104, sampling component 740 determines whether a sampling time has elapsed before taking readings from rear sensor 18 and front sensor 23. In one embodiment, a sampling counter (not shown) in memory 704 may be incremented each clock cycle and is reset in step 1106 after the sampling time has been reached or exceeded.

In step 1106, sampling component 740 turns on illumination LED 67 so that front sensor 23 and rear sensor 18 can read an optical reflectance measurement. In step 1108, sampling component 740 reads front sensor 23 and rear sensor 18, and stores the read measurements in sensor readings 713A and B. After reading or storing the measurements, sampling component 740 turns off illumination LED 67. Method 1100 may skip steps 1106 and 1110 to save on processing speed. However, more power may be required to keep LEDs 67 on longer.

At step 1112, sampling component 740 checks whether each of the front sensor 23 and rear sensor 18 readings are within a valid operating range as configured in operating parameters 712. For example, sampling component 740 may check whether all of the following conditions are met: front sensor reading > FRONT SENSOR MINIMUM, rear sensor reading > REAR SENSOR MINIMUM, front sensor reading < FRONT SENSOR MAXIMUM, and rear sensor reading < REAR SENSOR MAXIMUM. The uppercase variables indicate the minimum and maximum values of the front and rear sensor reading thresholds. In one embodiment, if one or more conditions fail, method 1100 proceeds to step 1116 and returns an error or an invalid result.

At step 1114, the sampling component 740 returns a valid calculated result, calculated using the measurement readings from the rear sensor 18 and the front sensor 23. For example, the sampling component 740 may calculate the result by using the following result equation:

result = [(Ri/R) — (Fi/F)] * SF

where,

Fi = initial reading of front sensor 23;

F = current reading of front sensor 23;

Ri = initial reading of rear sensor 18;

R = current reading of rear sensor 18; and

SF = scale factor, where scale factor is a large positive integer (e.g. 666) and is used to avoid values less than one in the calculated result.

FIG. 12 is a flow diagram of a method 1200. Method 1200 details the operation of step 1006 of method 1000, which determines whether a peak has been found. The peak may represent a maximum color intensity of the line formation zone 65 since most of the sample-conjugate fluid has flowed past the line formation zone 65 into the absorption pad 63. As described above, determining whether a pest, such as bed bugs, is present within the sample-conjugate fluid can be a time-critical process. A given test or profile evaluation result may only be valid within a specific time interval since the peak was detected.

In step 1202, the peak detection component 738 receives a calculated result (Calc) from the color checking component 736. Furthermore, during a first iteration of step 1202, a peak value (Peak) may be initialized to 0.

In step 1204, peak detection component 738 determines whether a peak has been found in a previous iteration of method 1200. During a first iteration of step 1204, method 1200 may proceed to step 1206.

At step 1206, the peak detection component 738 compares the calculated result to the Peak. If the calculated result exceeds the Peak, the method 1200 proceeds to step 1210, where the peak detection component 738 assigns the Peak to the current calculated result.

In step 1208, the peak detection component 738 detects whether a difference between the Peak and the calculated result exceeds a NOISE value, for example, 9. The peak detection component 738 may need to implement a noise filtering step such as step 1208 to eliminate a false positive peak detection, i.e., detect a peak that has not been reached.

At step 1212, if the difference between the Peak and the calculated result exceeds NOISE, then the peak detection component 738 may be more confident that a peak has been found. In subsequent iterations of the procedure 1200, step 1204 may indicate that a peak was found previously, i.e., in step 1212 of a previous iteration of the procedure 1200.

In step 1214, the peak detection component 738 returns whether a peak was found.

FIG. 13 is a flow diagram of a method 1300. Method 1300 details the operations of step 810 of method 800, which determines a test result or a pest profile assessment result. In step 1302, profile assessment component 742 initializes, resets, or clears counter 709.

In step 1304, the profile evaluation component 742 invokes the sampling component 740 to read measurements from the rear sensor 18 and the front sensor 23. The profile evaluation component 742 receives either a calculated result (Calc) or an indication that an error has occurred, i.e., that the calculated result is invalid. If an error occurs, the profile evaluation component 742 returns an invalid result in step 1306.

At step 1308, the profile evaluation component 742 determines whether the counter 709 has met or exceeded a TIME THRESHOLD parameter, such as the test time threshold 710, for the pest profile 706. The TIME THRESHOLD parameter is a predetermined period of time that is specific to a particular pest profile 706. If the test time threshold 710 has not been met, the method 1300 proceeds to step 1304, where the sensor values are resampled.

At step 1310, the profile evaluation component 742 determines whether the calculated result received from step 1304 has exceeded a MAX_THRESHOLD parameter, such as a maximum test intensity threshold 708 for the pest profile 706. If the calculated result exceeds the MAX_THRESHOLD, at step 1312, the profile evaluation component 742 determines that the pest profile 706 has been satisfied, and the presence of the pest associated with the pest profile 706 has been detected.

At step 1314, the profile evaluation component 742 determines whether the calculated result received from step 1304 falls below a MIN_THRESHOLD parameter, such as a minimum intensity threshold of the test threshold 708 for the pest profile 706. If the calculated result does not exceed or meet the MIN_THRESHOLD, at step 1316, the profile evaluation component 742 determines that the pest profile 706 was not satisfied, and an absence of the pest associated with the pest profile 706 was detected.

At step 1318, the profiling component 742 determines that because the calculated result falls between MIN_THRES<h>O<l>D and MAX_THRESHOLD, the result of the profiling evaluation is indeterminate. By returning an indeterminate output, even though the procedure 1300 does not always produce a definitive result, i.e., presence or absence detected, the procedure 1300 reduces false positives in pest detection. At step 1320, the determined result from step 1312, 1316, or 1318 is returned.

Although method 1300 has been described with respect to one pest profile, method 1300 may be adapted to determine whether multiple pest profiles (e.g., pest profile 706) are satisfied. Profile evaluation component 742 may be configured to perform method 1300 for each pest profile configured in pest profile 706, such that a profile evaluation result is determined for each configured profile.

Returning to FIG. 7, each component of components 730 to 742 is described below in turn:

The profile configuration component 730 configures the one or more pest profiles 706 to include specific thresholds for profile characteristics within the pest profiles 706. The thresholds may be provided, for example, by a manufacturer of the detection device 100. The profile configuration component 730 may be an optional component. In one aspect, the detection device 100 is simply preloaded or preconfigured with one or more pest profiles 706. Thus, the detection device 100 may run a single lateral flow assay test and detect the presence of more than one pest.

In one aspect, the sensing device 100 may include a communication interface (e.g., a USB port or a network port) for receiving new pest profiles 706 or updates to pest profiles 706. For example, the sensing device 100 may include a Bluetooth network chip (not shown) coupled to a network port of the processor 702 (i.e., the processor 21 of FIG. 1 ) such that a user or device manufacturer may add or program new profiles to the pest profiles 706, add profile features to existing pest profiles 706, or update one or more threshold values within the existing pest profiles 706. Thus, the pest profile 706 may be dynamically configured via the profile configuration component 730 to analyze a reagent band 16 currently incorporated into a specific lateral flow assay.

The power-on test component 732 may determine when to activate the processor 702 to begin detecting the presence of one or more pests. In one aspect, the power-on test component 732 may activate the processor 702 when an activation signal is detected. For example, an activation signal may be detected when the processor 702 is being powered (e.g., the power port 720 is receiving power) or when ambient light is being detected by at least one sensor (e.g., readings from the front sensor port 716 or the rear sensor port 718). In one embodiment, during startup of the processor 702, the power-on test component may initialize the ports 714 and registers or data within the memory 704.

Upon initialization, the power-on test component 732 may launch a self-test sequence to verify that the operating parameters 712 are met. For example, the power-on test component 732 may cycle through each LED, controlled by the processor 702, to determine whether each of the LEDs is operating. In one aspect, upon power-up or initialization, the self-test sequence may include determining whether a detected ambient light is above a threshold stored in the operating parameters 712. The power-on test component 712 may control one or more LEDs to flash or illuminate to indicate the current progress or result of the self-test. Upon completion of the self-test, the sampling component 740 may be requested to take an initial reading from the front sensor 23 and an initial reading from the rear sensor 18.

The liquid monitoring component 734 may detect whether a lateral flow assay test is being performed on the reagent strip 16 of FIG. 2 based on measurements read at the front sensor port 716 or the rear sensor port 718. As described with respect to FIG. 2 , a sensor reading may be a voltage value representative of an optical reflectance of incident light, e.g., color intensity, detected by the sensor. In one embodiment, the liquid monitoring component 734 may call or invoke the sampling component 740. The sampling component 740 reads from one or more sensors to obtain measurement readings used to calculate a result used by the liquid monitoring component 734 to calibrate the front sensor 23 and the rear sensor 18, as explained above with respect to FIG. 9 .

In a typical operation of the lateral flow assay test on test strip 16, sample fluid may flow from wick 56, through reagent pad 61, and past line formation zone 65 to absorption pad 63. In one embodiment, liquid monitoring component 734 receives a calculated result from sampling component 740. Sampling component 734 may then compare the calculated result to a liquid threshold in operating parameters 712 before determining to proceed with the detection algorithm or process. When the calculated result exceeds the liquid threshold, at least a portion of the liquid in the sample has reached the portion of the reagent strip 16 opposite the front sensor 23 or the rear sensor 18. The liquid verification component 734 may further use the received calculated result to calibrate the initial readings of the front and rear sensors (e.g., reset the initial sensor readings) if a current front sensor reading deviates from the previous initial front reading and the calculated result is close to 0, indicating that no liquid is being detected.

The color checking component 736 may detect whether a sufficient amount of color particles within the reagent pad 61 have flowed past the line forming zone 65 opposite the front sensor 23. In one aspect, if not enough color particles migrate or flow past the line forming zone 65, not enough sample-conjugate molecules may be captured. As a result, the processor 702 may detect a false negative (i.e., detect an absence of one or more insect pests even though an insect pest could be present). The color checking component 736 may invoke or call the sampling component 740. The sampling component 740 reads from the front sensor 23 and the rear sensor 18 to obtain sensor readings. The readings are necessary for the color checking component 736 to determine whether a sufficient amount of color particles have been detected. In one aspect, the color check component 736 may be called to begin execution when the liquid check component 734 detects sample fluid.

In a typical lateral flow assay test operation, as more sample-conjugate fluid flows past the line formation zone 65, colored particles (e.g., colloidal gold) within the sample-conjugate fluid may intensify the coloration detected by the front sensor 23 opposite the line formation zone 65. As most of the sample-conjugate fluid has migrated to the absorbent pad 63, fewer and fewer colored particles passing through the line formation zone 65 may be detected. The color check component 736 may use a calculated result from the invoked sampling component 740 to determine a peak in intensity in the coloration, indicating that most of the sample-conjugate fluid has migrated past the line formation zone 65. The color check component 736 may invoke or call the peak detection component 736 to perform peak detection.

The sampling component 740 may verify that each of the front sensors 23 and rear sensors 18 are operating properly before reading from the sensors to calculate a result. In one embodiment, after a sampling delay, the sampling component 740 may sample and store the readings from the front sensor 23 and the rear sensor 18 in sensor readings 713A and B, respectively. The sampling component 740 may also store a calculated result in memory 704. In one aspect, the sampling component 740 may determine whether a sensor is operating properly by verifying that the value of the sensor reading is between operating thresholds for the sensor defined in operating parameters 712.

In one aspect, the sampling component 740 may calculate a result according to the following equation:

result = [(Ri/R) — (Fi/F)] * SF

where,

Fi = initial reading of front sensor 23;

F = current reading of front sensor 23;

Ri = initial reading of rear sensor 18;

R = current reading of rear sensor 18; and

SF = scale factor, where scale factor is a large positive integer (e.g. 666) and is used to avoid values less than one in the calculated result.

In one aspect, the provided equation “result = [(Ri/R) — (Fi/F)] * SF” may be used by the liquid check component 734, the color check component 736, as well as the profile evaluation component 742.

In one aspect, by taking into account the relationship between the initial and current readings of the front sensor 23 and the rear sensor 18 prior to testing, the calculated result takes into account environmental conditions to provide a more accurate test result. Aspects describing the use and advantages provided by the above equation are described in U.S. Patent No. 7,499,170 B2 entitled “System and Method for Reading a Test Strip”. Depending on the type of sensors being used, the calculated result may utilize a modified equation. For example, in one aspect where a sensor is a photodiode, the following equation may be used instead: result = [(R/Ri) — (F/Fi)] * SF.

For example, during initialization and prior to testing, both the front sensor 23 and the rear sensor 18 may provide a proportional response or measurement readings, and both sensors may track similarly, because both sensors are exposed to the same environmental conditions and both sensors are exposed to a similar light source, i.e., the illuminated LED 67. To clarify what is meant by the phrases “proportional response” and “track similarly,” suppose that both sensors are exposed to a light source of a given wavelength and intensity. Also suppose that the distance between both sensors and the light source is similar. The light would cause each sensor to produce an output response or measurement reading within the particular operating range of each sensor. One output response could be greater or less than the other output response without affecting the test result. Now take the same two sensors and expose them to a light source of the same wavelength but at a lower intensity. The output response of each sensor may be lower than the previous output response, meaning they remain similar.

The profile evaluation component 742 may compare or match a calculated or stored result to the pest profile 706 to determine whether one or more pest profiles within the pest profile 706 have been satisfied. The profile evaluation component 742 may call or invoke the sampling component 740 to calculate or obtain the calculated result. In one aspect, for each profile configured in the pest profile 706, the profile evaluation component 742 may wait a minimum time within the test time threshold 710 before comparing the calculated result to each profile. This produces a profile evaluation result for each insect pest. The time delay may be necessary to allow enough conjugate molecules in the sample to react with the antibodies immobilized in the line formation zone 65 such that the profile evaluation component 742 can determine a valid result.

In one aspect, the profile evaluation component 742 may compare the calculated result to, for example, a maximum intensity threshold within the pest profile 706. This determines whether the presence of an insect pest associated with the pest profile 706 is detected. Other comparisons may be made based on characteristics of the profile stored in the pest profiles 708. For example, a characteristic of the profile may include a rate of change in light intensity.

In determining whether a pest profile of the pest profiles 706 has been met, the profile evaluation component 742 may cause the output ports 714 to turn on one or more LEDs. This indicates a profile evaluation result. For example, the profile evaluation component 742 may energize or turn on the red LED 32 to indicate the detected presence of bed bugs. That is, the profile evaluation result is positive, indicating that the bed bug pest profile 506 has been met. Conversely, the profile evaluation component 742 may turn on the green LED 29 when it detects the absence of bed bugs, that is, the profile evaluation result is negative. In one embodiment, the profile evaluation component 742 may output a result (e.g., by turning on both the green LED 29 and the red LED 32) to indicate that the profile evaluation result was indeterminate.

In one aspect, the profile evaluation component 742 may indicate a profile evaluation result for each configured pest profile within the pest profile 706. For example, the results display 25 may include an LED-based device for each configured profile that is controlled to illuminate or change to a specific color to indicate each specific profile evaluation result. In one embodiment, the profile evaluation component 742 may display a severity of the detected presence of an insect pest based on an intensity of the coloration of the line formation zone 65.

FIG. 14 is a diagram illustrating example results obtained for lateral flow assay tests of samples analyzed by components of detection device 100 of FIG. 1. As shown, the table of infestation intensity values on the left shows calculated results for six lateral flow assay tests of samples performed on the same detection device, such as detection device 100 of FIG. 1. For each assay test, sampling component 740 may store calculated results at three sampling times: 3min, 5min, and 10min.

The pest profile 706 may include a bed bug profile that includes, for example, a minimum intensity threshold of 9, a maximum intensity threshold of 15, and a test time threshold between 4 and 6 minutes. For example, the test time threshold may be 5 minutes. This test time threshold is a predetermined time specific to the bed bug profile. The test results for Test 1 through Test 3 exceed the maximum intensity threshold at the 5-minute mark. The test result for Test 6 falls between the minimum and maximum intensity thresholds. And, the calculated test result for Test 5 falls below the minimum intensity threshold. Thus, the profile assessment component 742 may control the LEDs to indicate a presence of bed bugs for test tests 1 through 3, an absence of bed bugs for test test 5, and an indeterminate result for test test 6. In one aspect, a bed bug profile may include severity thresholds such that the profile assessment results for test tests 1 through 3 indicate medium severity, low severity, and high severity, respectively. One or more LEDs may be controlled to indicate the severity of the infestation levels by, for example, turning on, flashing at a specific rate, or emitting light at a specific intensity.

In one aspect, pest profile 706 may include the described bed bug profile and a cockroach profile that includes, for example, a minimum intensity threshold of 11, a maximum intensity threshold of 14, and a test time threshold between 9 and 11 minutes. Although at the 10 minute sampling time, test tests 1 through 3 and 6 may each exceed the maximum intensity threshold of 14, profile evaluation component 742 may only indicate a detected presence of cockroaches for test test 6. Therefore, processor 702 may be a prioritized insect pest detector. The types of pests possibly detected may be prioritized based on the test time thresholds, from the shortest delay to the longest. For example, processor 702 may be configured to first detect for bed bugs at the 5 minute sampling time before second detecting for cockroaches at the 10 minute sampling time.

To test the effectiveness of the detection device 100 of FIG. 1, having the processor 702 of FIG. 7 and operated in accordance with the methods of FIGS. 8-13, 17 bed bug infested beds were examined. In this field test, each of the 17 beds was visually confirmed to have at least 10 live bed bugs. In this field test example, a fluid sample was created for each of the 17 beds and applied to the detection device 100 to detect past or present bed bug infestation. Briefly, as described with respect to FIG. 1, the sample fluid was created by first using a swab material and a standardized collection procedure to swab each bed and then dipping the swab material into an extract buffer.

In this field test example, the bed bug detection result for each of the 17 beds was generated and displayed after 10 minutes. As discussed above with respect to pest profile 706, this 10 minute test time is an example of a specific time period for bed bugs. Implementing the procedures of FIGS. 8 through 13, the detection device 100 detected a prior or present bed bug infestation in 13 of the 17 bed bug infested beds. Thus, this field test example shows that by using the detection device 100, a pest control operator can determine bed bug infestations on-site and in a short period of time, e.g., within 10 minutes, with high accuracy. The pest control operator would not have to rely on current detection procedures, such as obtaining a sample of suspected bed bug residue to be analyzed at an outside testing laboratory, which often requires 24 to 48 hours to produce a test result.

Any of the above aspects may include the following anti-bed bug monoclonal antibodies or antigen-binding fragments thereof, mutants, conjugated antibodies or conjugated antigen-binding fragments, compositions, kits, hybridomas, polynucleotides, polypeptides, vectors, cells, or methods, as disclosed in U.S. Provisional Patent Application No. 62/244,189 filed on October 21, 2015, entitled “Anti-bed bug monoclonal antibodies and methods of making and uses thereof”.

Terminology

As used herein, the term “bed bug” refers to any species or strain of Cimex.

The terms “antibody” and “antibodies” are terms of art and may be used interchangeably herein to refer to a molecule or molecules with an antigen binding site that specifically binds to an antigen.

The term “monoclonal antibody” refers to a homogeneous population of antibodies that engage in the specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies which typically include different antibodies directed against different antigenic determinants. Additionally, “monoclonal antibody” refers to such antibodies manufactured in any manner, including, but not limited to, by hybridoma, phage selection, recombinant display, and transgenic animals. The term “antigen-binding fragment” refers to a portion of an antibody that is capable of specifically binding an antigen. Examples of antibody fragments include, but are not limited to, heavy chain variable region fragments, light chain variable region fragments, Fab, Fab', F(ab')2, scFv fragments, Fv fragments, linear antibodies, single chain antibodies, multispecific antibodies, minibodies, dibodies, triabodies, and tetrabodies.

The terms “variable region” or “variable domain” are terms of the art and may be used interchangeably herein to refer to a portion of an antibody that differs widely in sequence between antibodies and is used in the binding and specificity of a particular antibody for its particular antigen. Each of the heavy and light chain variable regions consists of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs of each chain contribute to the formation of the antigen binding site of the antibodies.

The term “specifically binds” refers to molecules that bind to an antigen (e.g., an epitope or immune complex) as understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides, generally with lower affinity, as determined, for example, by immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art.

As used herein, the term “detect” encompasses both quantitative and qualitative detection.

As used herein, the term “effective amount” refers to the amount that achieves a desired effect.

As used herein, the terms “host cell” and “cell” may be used interchangeably and may refer to any type of cell, for example, a primary cell, a cell in culture, or a cell of a cell line, including a hybridoma.

An antibody, antigen-binding fragment, host cell, and cell, as referred to herein, include “isolated” forms that have been separated or recovered from a component of its native environment, such as by the separation or removal of contaminants that would interfere with the uses of the antibody, antigen-binding fragment, host cell, or cell, where such contaminants may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials.

Certain aspects may include an antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-122644, or an antigen-binding fragment thereof. The bed bug monoclonal antibody designated herein as BB2 is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644.

Certain aspects may include an antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645, or an antigen-binding fragment thereof. The anti-bed bug monoclonal antibody designated herein as BB7 is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645.

Certain aspects may include a monoclonal antibody or antigen-binding fragment thereof comprising the heavy chain and light chain complementarity determining regions (CDRs) of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7] (see, e.g., the discussion of CDRs in Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983), and Chothia et al., J. Mol. Biol. 196:901a 917 (1987)). Methods for determining CDRs are well known, including an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest,(5th ed.), and Chothia et al., J. Mol. Biol. 196:901a 917 (1987)). ed., 1991, National Institutes of Health, Bethesda, MD)), and an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., J. Molec. Biol.273:927a 948 (1997)). Furthermore, combinations of these two approaches can be used to determine CDRs. CDRs can also be determined according to Lefranc M-P,The Immunologist7: 132 to 136 (1999) Lefranc M-P,et al., Nucleic Acids Res27: 209 to 212 (1999) MacCallum RMet al., J. Mol. Biol. 262: 732 to 745 (1996) Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains”, in Antibody Engineering, Kontermann and Dübel, eds., chapter 31, pp. 422 to 439, Springer-Verlag, Berlin (2001); and the AbM antibody modeling software from Oxford Molecular (Oxford Molecular Group, Inc.).

Certain aspects may include an antibody or antigen-binding fragment thereof comprising the heavy and light chain variable regions of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. Typically, a variable region is located about 110 to 120 amino acids from the amino terminal in the mature heavy chain and about 90 to 115 amino acids from the amino terminal in the mature light chain.

Certain aspects may include an antibody or antigen-binding fragment thereof comprising the heavy and light chains of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. Each heavy chain comprises a heavy chain variable region and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain comprises a light chain variable region and a light chain constant region. The light chain constant region comprises a (CL1) domain.

Monoclonal antibodies included in the disclosure may be, but are not limited to, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies such as bispecific antibodies, fusion proteins comprising an antigen-determining portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired activity. The antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), of any class (e.g., IgG-i, IgG<2>, IgG<3>, IgG<4>, IgA<1>, or IgA<2>), or of any subclass (e.g., IgG<2>a or IgG<2>b) of immunoglobulin molecule. The techniques for producing antibodies will be apparent to skilled practitioners. Monoclonal antibodies may be prepared, for example, by the use of hybridoma procedures such as those described by Kohler and Milstein, Nature 256:495 (1975). By the hybridoma procedure, a mouse, hamster, or other appropriate host animal is immunized to elicit production by the lymphocytes of antibodies that will bind specifically to an immunizing antigen. Lymphocytes may also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line by the use of, for example, polyethylene glycol, to form hybridoma cells which can then be selected from unfused lymphocytes and myeloma cells.

Monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Patent 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, such as by RT-PC, using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined by conventional methods. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which allow for the generation of monoclonal antibodies when transfected into host cells, including, but not limited to, E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. Recombinant monoclonal antibodies or fragments thereof of the desired species can also be isolated from phage display libraries expressing CDRs of the desired species (see, for example, McCafferty et al., Nature 348:552 to 554 (1990) Clackson et al., Nature 352:624 to 628 (1991); and Mark et al., J. Mol. Biol. 222:581 to 597 (1991)).

Antigen-binding fragments in embodiments may be produced by any known method and include a portion of an antibody that is capable of specifically binding an antigen. Examples of antibody fragments include, but are not limited to, heavy chain variable region fragments, light chain variable region fragments, Fab, Fab', F(ab')2, scFv fragments, Fv fragments, linear antibodies, single chain antibodies, multispecific antibodies, minibodies, diabodies, triabodies, and tetrabodies (see, e.g., Hudson and Souriau, Nature Med. 9:129-134 (2003) and U.S. Patent 5,641,870). Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1993) Brennan et al., Science 229:81 (1985)). In certain embodiments, the antibody fragments are produced recombinantly. For example, the antibody fragments can be expressed and secreted from E. coli or other host cells, thereby allowing the production of large quantities of the fragments. Such antibody fragments can also be isolated from antibody phage libraries. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

In certain aspects, any of the antibodies or antigen-binding fragments thereof may be capable of binding to a bed bug antigen in a whole bed bug lysate or a harvest paper extract comprising bed bug waste material. Methods of producing whole bed bug lysates and harvest paper extracts comprising bed bug waste material will be apparent to those skilled in the art based on known extraction techniques and the methods disclosed herein. Whole bed bugs including nymphs, males, and/or females may be obtained from an infestation area or from an experimentally or commercially maintained bed bug colony, and may include any Cimex species or strain, including, but not limited to, Cimex lectularius and the Harlan strain. Collection paper containing bed bug waste material may include bed bug frass and/or tissues, for example, and can be obtained from commercial sources (e.g., i2LResearch USA Inc., Baltimore, Maryland, USA).

The disclosure may include a mutant of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. Mutants may contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, conservative substitution refers to the replacement of one amino acid for another within the same general class such as, for example, one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art. Mutations may also include deletions, insertions, inversions, and repeats. Mutations may be introduced by general molecular biology procedures known in the art, including, but not limited to, error-prone PCR, oligonucleotide-directed mutagenesis, site-directed mutagenesis, and heavy or light chain shuffling.

The disclosure may include a monoclonal antibody or antigen-binding fragment thereof that has one or more characteristics that are substantially similar to those of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. The phrase “substantially similar,” as used herein, denotes a high enough degree of similarity between two characteristics that those skilled in the art would consider the difference to have little or no biological and/or statistical significance. In certain embodiments, the difference between two numerical values may be less than about 15%, 10%, 5%, 2%, or 1%. Characteristics of the deposited antibodies may include one or more properties, such as, but not limited to, binding specificity (e.g., Kd value), antigenic/epitope determinants, and polynucleotide or polypeptide sequences. In certain aspects, the monoclonal antibody or antigen-binding fragment thereof has a polynucleotide or polypeptide sequence that is at least 90% to 99%, at least 95% to 99%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the polynucleotide or polypeptide sequence of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the monoclonal antibody or antigen-binding fragment thereof has one or more of the same characteristics as the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the monoclonal antibody or antigen-binding fragment thereof binds to the same antigenic determinant/epitope as the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7].

The terms “epitope” or “antigenic determinant” may be used interchangeably herein to refer to the portion of an antigen capable of being specifically recognized and bound by a particular antibody. An epitope typically includes at least 3, and more typically, at least 5 or 8 to 10 amino acids in a unique spatial conformation. An epitope may be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope may, for example, arise from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). The epitope to which an antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled to mass spectrometry (e.g., electrospray liquid chromatography mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be carried out using any of the methods known in the art (e.g., Giegé Ret al.,(1994)Acta Crystallogr D Biol Crystallogr50(Pt 4):339-350 McPherson A (1990)Eur J Biochem189:1-23 Chayen NE (1997) Structure 5:1269-1274 McPherson A (1976)J Biol Chem251:6300-6303). Antibody:antigen crystals can be studied by well-known X-ray diffraction techniques and can be refined by the use of computer programs such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymol (1985) volumes 114 and 115, eds Wyckoff H Wet al., U.S. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37 to 60 Bricogne G (1997) Meth Enzymol 276 A: 361 to 423, ed Carter C W; Roversi Pet al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316 to 1323). Mutagenesis mapping studies may be carried out using any method known to those skilled in the art. See, for example, Champe Met al.,(1995)J Biol Chem270:1388 to 1394 and Cunningham BC & Wells JA (1989)Science244:1081 to 1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

The disclosure may include a conjugated monoclonal antibody or conjugated antigen-binding fragment comprising any of the antibodies, antigen-binding fragments, or mutants described herein and a detection agent, including the detection agents described above. The detection agent may be directly or indirectly conjugated to the antibody, antigen-binding fragment, or mutant. The detection agent may be detectable itself or, in the case of an enzymatic label, may catalyze the chemical alteration of a substrate compound or composition that is detectable. The detection agent includes, but is not limited to, a radiolabel, a fluorophore, a chromophore, an imaging agent, or a metal, including a metal ion. In certain aspects, the detection agent is colloidal gold or gold nanoparticles. In certain embodiments, the colloidal gold or gold nanoparticles are composed of gold particles having a size of 1 to 300 nm, 1 to 250 nm, 10 to 200 nm, 20 to 150 nm, 20 to 100 nm, 20 to 80 nm, 20 to 60 nm, 20 to 50 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm. In certain aspects, the conjugated antibody or antigen-binding fragment conjugate comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], or an antigen-binding fragment thereof, or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], or an antigen-binding fragment thereof.

The disclosure may include a composition comprising any of the antibodies or antigen-binding fragments thereof, mutants, or conjugated antibodies or antigen-binding fragments conjugated herein, or a combination thereof. In certain aspects, the composition comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain embodiments, the composition comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], and a conjugated antibody comprising the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7] and a detection agent. In certain aspects, the composition comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], and an antibody conjugate comprising the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and a detection agent.

The disclosure may include a kit comprising any of the antibodies or antigen-binding fragments thereof, mutants, conjugated antibodies or conjugated antigen-binding fragments, or compositions herein, or a combination thereof. In certain aspects, a kit comprises at least one component in one or more containers. In some aspects, the kit comprises components necessary and/or sufficient to carry out a detection assay, including controls, instructions for carrying out the assays, any necessary devices, and/or software for analysis and presentation of results. Suitable devices include those disclosed in U.S. Patent No.

7,220,597 and 7,214,542.

The disclosure may include a hybridoma capable of producing an antibody, wherein the hybridoma is deposited with the ATCC under Accession Number PTA-122644 [BB2] or wherein the hybridoma is deposited with the ATCC under Accession Number PTA-122645 [BB7].

The disclosure may include an isolated polypeptide comprising an amino acid sequence at least 90% to 99%, at least 95% to 99%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of a heavy or light chain variable region or a heavy or light chain, of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the polypeptide comprises the amino acid sequences of the CDRs of a heavy or light chain variable region of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the polypeptide comprises the amino acid sequences of the heavy or light chain variable region, or of either the heavy or light chain, of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [B<b>7].

The disclosure may include an isolated polynucleotide comprising a nucleic acid sequence at least 90% to 99%, at least 95% to 99%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleic acid sequence encoding a heavy or light chain variable region, or a heavy or light chain, of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the polynucleotide comprises nucleic acid sequences encoding the CDRs of a heavy or light chain variable region of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7]. In certain aspects, the polynucleotide comprises a nucleic acid sequence encoding the heavy or light chain variable region, or the heavy or light chain, of the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7].

Embodiments may include a vector comprising one or more of the isolated polynucleotides of the invention. In certain embodiments, the vector is an expression vector.

The disclosure may include an isolated cell that produces an antibody, antigen-binding fragment, or mutant of the invention. In certain embodiments, the cell is a hybridoma. In certain embodiments, the cell comprises one or more vectors of the invention. In certain aspects, the cell comprises one or more polynucleotides of the invention.

The disclosure may include a method of making an antibody, antigen-binding fragment, or mutant of the invention, comprising culturing an isolated cell that produces the antibody, antigen-binding fragment, or mutant, and isolating the antibody, antigen-binding fragment, or mutant from the cultured cell.

Cells include, but are not limited to, hybridomas, prokaryotes, yeast, insects, or higher eukaryotic cells. Hybridomas producing monoclonal antibodies can be propagated in culture in vitro using standard procedures (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. Prokaryotes include Gram-negative or Gram-positive organisms, e.g. E. coli bacilli. Higher eukaryotic cells include, but are not limited to, established cell lines of mammalian origin. Examples of suitable mammalian cell lines are COS-7, L, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. In certain aspects, any of the antibodies, antigen-binding fragments or mutants of the invention are produced by isolated cells following transfection of the cells with vectors comprising polynucleotides encoding the antibody, antigen-binding fragment or mutant sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cellular hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Mammalian expression vectors may comprise non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, donor and acceptor splice sites, and transcriptional termination sequences. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

Antibodies, antigen-binding fragments, or mutants may be isolated from cells or from the culture medium, or from ascites fluid for in vivo propagation of hybridomas. Isolation of antibodies, antigen-binding fragments, or mutants may be carried out according to any suitable procedure. Such standard procedures include chromatography (e.g., ion exchange, affinity and size column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Procedures known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publication Nos.

2008/0312425, 2008/0177048 and 2009/0187005.

The disclosure may include a method of detecting bed bugs, comprising contacting a sample comprising a bed bug antigen with any of the antibodies, antigen-binding fragments, mutants, antibody conjugates or antigen-binding conjugate fragments, or compositions, or a combination thereof, and detecting binding of the bed bug antigen to the antibody or antigen-binding fragment, mutant, antibody conjugate or antigen-binding conjugate fragment, composition, or combination thereof. “A sample” includes, but is not limited to, whole bed bugs, bed bug parts, bed bug litter material, lysates or extracts thereof, extracts of collection paper comprising bed bug litter material, and fluids containing the same. The contacting may be by any suitable method. In certain aspects, contacting is accomplished by applying a sample comprising the bed bug antigen to an antibody, antigen-binding fragment, mutant, conjugated antibody or conjugated antigen-binding fragment, or composition of the invention, or a combination thereof that is immobilized or otherwise positioned on a surface. Any acceptable surface may be used, as will be appreciated by those skilled in the art, including, but not limited to, a nitrocellulose membrane or a pad composed of a suitable material, and may include a sandwich, well, or lateral flow design. The sample is contacted with an antibody and a conjugated antibody. In certain aspects, the antibody is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], and the conjugated antibody comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7] and a detection agent. In certain aspects, the antibody is produced by the hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], and the conjugated antibody comprises the antibody produced by the hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] and a detection agent. In certain aspects, contacting further comprises contacting the antibody, antigen-binding fragment, mutant, conjugated antibody, or conjugated antigen-binding fragment, or composition, or a combination thereof with a control sample for comparison to the test sample.

Detection may be by any suitable method and may include quantitative or qualitative detection. Such methods include, but are not limited to, antigen binding assays that are well known in the art, such as lateral flow assays, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich assays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC). Detection may include visual analysis of a colorimetric, fluorescent, or luminescent reaction, for example, or may include the use of a device that measures such reactions. Suitable devices include those disclosed in U.S. Patent No.

7,220,597 and 7,214,542, as well as a device disclosed herein. In certain embodiments, the detection comprises performing a lateral flow assay. In certain aspects, the detection occurs in 1 to 20 minutes, 1 to 15 minutes, 1 to 10 minutes, 1 to 5 minutes, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, within 20 minutes, within 15 minutes, within 10 minutes, or within 5 minutes.

The amount of the antibody, antigen-binding fragment, mutant, conjugated antibody or antigen-binding fragment, composition, or combination thereof, may include any effective amount, which will be apparent to those skilled in the art based on known detection methods and the methods disclosed in the examples. The sample may be diluted or undiluted.

In certain aspects, the method further comprises collecting a sample comprising the bed bug antigen with a collection device and extracting antigen from the sample. The sample may be collected from any surface associated with bed bug infestation, including, but not limited to, bedding, mattresses, upholstery, carpets, rugs, and furniture. The collection device may be any suitable device, including, but not limited to, a swab, such as a cotton swab, a vacuum cleaner, or any material that can be used to collect debris, including, but not limited to, a wipe, tissue, or towel. In certain aspects, the collection device is a swab. In certain aspects, extracting antigen from the sample comprises solubilizing antigen in the sample with an extraction buffer. Suitable extraction buffers will be apparent to those skilled in the art in view of the well-known extraction buffers and those disclosed in the examples.

The properties of the BB2 and BB7 antibodies are described in the following examples, which are offered by way of illustration and not limitation.

Example 1: Generation of monoclonal antibodies against bed bugs

Mice were immunized with whole bed bug lysates and bed bug paper extracts (i.e., extracts from bed bug collection paper containing waste material from a bed bug colony).

Whole bed bug lysates were produced from nymphs, males, and females of a bed bug colony (Harlan strain, i2LResearch USA Inc., Baltimore, Maryland, USA) that were frozen and ground in 1X phosphate-buffered saline (PBS). Lysates were clarified through a 0.45-micron syringe filter. Protein concentration was determined by a standard Bradford protein assay. Clarified and quantified extracts were aliquoted into 1.5 mL Eppendorf tubes and stored at -80 °C.

Bed bug collection paper (i2LResearch USA Inc., Baltimore, Maryland, USA) was cut into pieces of approximately 1 cm2 and placed in 2 mL plastic centrifuge tubes. Extraction was carried out by adding 1.0 mL of 50 mM PBS (pH 7.4) and mixing the tubes for 30 min on a tube rocker. After 30 min, the extract was completely extracted by passing the mixture through a 5 mL syringe. This collected extract was then used to obtain more extract from fresh collection paper by serially adding the extract to the freshly cut paper and repeating the process. The final extract was then centrifuged at 12,000 rpm for 10 min to remove particulate matter. The supernatant was removed and saved, and the pelleted material was discarded. The protein concentration of the supernatant was determined by Bradford assay as 0.6 mg/mL. The final solution (i.e., “bed bug paper extract”) was stored at -20 °C.

Four 4- to 5-week-old Balb/c mice (Harlan Laboratories, Inc., Indianapolis, Indiana, USA) were immunized subcutaneously in the back with 50 µg of whole bed bug lysate mixed with 100 µl of adjuvant. The adjuvant used for two of the mice was a traditional adjuvant (Freund's Adjuvant, Sigma-Aldrich Co. LLC, St. Louis, Missouri, USA) and the adjuvant used for the remaining two mice was a water-soluble adjuvant (ImmuQuik®, KCH Scientific, San Jose, California, USA).

On Day 14 after the initial immunization, immunized mice were boosted with 50 µg of whole bed bug lysate mixed with 100 µl of an adjuvant as originally used for each mouse. On Day 37, mouse sera were titered by standard enzyme immunoassay, using horseradish peroxidase-conjugated goat anti-mouse antibody as the secondary antibody/enzyme conjugate and 3,3',5,5'-Tetramethylbenzidine as the chromogenic substrate, and 10 µg/ml of whole bed bug lysate as the antigen source. The two mice immunized with whole bed bug lysate in the water-soluble adjuvant produced higher titers. However, since titers were not strong overall, all four immunized mice were boosted with twice the amount of whole bed bug lysate (i.e., 100 µg) on Day 51 and then again on Day 78. On Day 107, all four mice were boosted with 15 µl of bed bug paper extract. On Day 117, the mouse with the highest previous titer was titer challenged by using either whole bed bug lysate or bed bug paper extract as the antigen source. A weak reaction was observed for bed bug paper extract. On Day 134, all four mice were boosted with 100 µl of bed bug paper extract by using ImmuQuik® as an adjuvant. On Day 151, mice were titer challenged using bed bug paper extract as the antigen source, and the mouse with the highest titer was boosted with 100 µl of bed bug paper extract.

Spleen cells were harvested from the two highest titer mice, one on Day 154 and the other on Day 216, and fused with SP 2/0 murine myeloma cells using polyethylene glycol. The fused cells were cultured in selection medium for 10 days, followed by screening with bed bug paper extract as an antigen source. Approximately 41 positive clones were identified in the primary screen, and approximately 25 positive clones were confirmed in the secondary screen. Stable cell lines were subcloned, ascites were produced for more than 25 clones, and antibodies were purified (e.g., in amounts of 2 to 5 mg). Two antibodies, referred to herein as BB2 and BB7, were determined by enzyme immunoassay to be highly reactive with bed bug paper extract compared to antibodies from other clones and were selected for further study. Hybridomas producing antibodies BB2 and BB7 were deposited under the Budapest Treaty with the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209, on October 8, 2015, and given ATCC Accession Nos. PTA-122644 and ATCC PTA-122645, respectively.

Example 2: Sandwich capture assay of monoclonal antibodies against bed bugs

A sandwich capture assay was performed using BB2 or BB7 antibody as the capture antibody and gold-conjugated BB2 or gold-conjugated BB7 as the detector antibody. The bed bug paper extract described in Example 1 was used as the source of bed bug antigen. A rabbit polyclonal antibody against bed bug as described in US Publication No. 2015 0064727 was used as the positive control capture antibody and PBS was used as the negative antigen control.

Capture antibodies at concentrations of 2.0 mg/ml were spotted as 0.3 µl spots on nitrocellulose paper bands. Bed bug paper extract was added to the reactive bands and PBS was added to the negative control bands. Gold-conjugated BB2 antibody (at pH 7 or 9) or gold-conjugated BB7 antibody (at pH 9) as a detector antibody was added to the bands as shown in FIG. 15. The negative control bands showed an absence of binding by the detector antibodies. The reactive bands showed a red staining of the capture spots indicating binding of the gold-conjugated detector antibodies to the bed bug antigen immobilized on nitrocellulose by the capture antibodies. As summarized in Table 1, strong reactions were observed with the use of either BB2 or bB7 antibody as the capture antibody. In contrast, very weak (i.e., “+”) or uncertain (i.e., “+/-”) reactions were observed using the rabbit polyclonal antibody against bed bugs as the capture antibody.

Table 1: Intensities Observed in the Sandwich Capture Assay

Capture antibody

Example 3: Lateral flow immunoassay for detecting bed bug antigens

An example of a lateral flow immunoassay was designed to detect bed bug antigens from swab samples taken in areas with different levels of bed bug infestation. Extraction buffers were prepared and tested for efficient extraction of bed bug antigen from swabs, adequate flow on nitrocellulose reagent bands, and non-specific or low binding. Swab samples were extracted and serially diluted to investigate the sensitivity of the assay. The precision of the assay was investigated by testing replicates and reading signal intensities using a reagent band reader.

Preparation of nitrocellulose membrane: Nitrocellulose membranes (CN 140, 25 mm, Sartorius Corp., Bohemia, New York, USA) were sprayed with 1.0 mg/mL of bed bug antibody BB7 as a test line and 0.5 mg/mL of goat anti-mouse antibody (Lampire Biological Laboratories, Pipersville, Pennsylvania, USA) as a control line by using a Biodot Air Jet (Biodot, Irvine, California, USA) to strip the nitrocellulose membranes. The Stripping Buffer was IX PBS, 0.2% Sucrose, pH 7.4. The test line and control line were sprayed 7 mm apart, with the test line located 10 mm from the bottom of the membrane. The membranes were stripped at a rate of 1.0 pl/cm. Membranes were dried at 37 °C for 1 hour and stored in a dried aluminum foil bag. The stripped membranes were kept desiccated overnight before blocking.

Nitrocellulose membrane blocking: After drying overnight, the stripped membranes were placed in blocking solution (25 mM KPO<4>, 0.2% Casein, 0.5% Boric Acid, 0.02% Sucrose, 0.1% Surfactant 10G, 0.5% PVA) with the orientation of the test line at the bottom of the nitrocellulose and the control line at the top of the nitrocellulose. The blocking solution was allowed to reach the top of the membrane. The membranes were removed from the blocking solution and placed on a rack to dry at 37 °C for 1 hour. The blocked membranes were kept desiccated in a plastic bag and stored in a dry room.

Gold conjugation to antibodies: A Slide-A-Lyzer™ desalting cassette (molecular weight cutoff 10000, Thermo Fisher Scientific Inc., Carlsbad, California, USA) was used to dialyze anti-bed bug BB2 antibody overnight into 10 mM KPO<4>, pH 7.4. The final concentration of BB2 antibody after dialyzation was 0.875 mg/ml. A colloidal gold solution containing 40 nm particles and an optical density (OD) of 2.28 at 525 nm was adjusted at room temperature to pH 8.6 with freshly made 0.1 M K<2>CO<3>. The dialyzed BB2 antibody was added to the colloidal gold solution while shaking. The solution was incubated for 30 min on a rotator at room temperature. The conjugate was blocked with 10 µl (per 1 ml OD 2 colloidal gold) of gold conjugate blocking buffer (25 mM KPO<4>, 0.2% Bioterge, 6% BSA, 0.3% Sucrose) on a rotator at room temperature for 10 minutes. The gold conjugate was centrifuged at 12000 RPM, 4 °C for 20 minutes and the supernatant was discarded. The conjugate pellet was resuspended with 0.2 ml (per 1 ml OD 2 colloidal gold) of resuspension buffer (1:5 dilution of gold conjugate blocking buffer in 25 mM KPO<4>, 0.05% sodium azide). The Do of the gold-conjugated BB2 antibody was checked with a spectrophotometer and adjusted to 10. The gold-conjugated BB2 antibody was stored at 4 °C.

Gold conjugate pad preparation: A P-1000 pipette was used to saturate 300 mm Ahlstrom 8950 conjugated glass fiber pads (Ahlstrom, Helsinki, Finland) with blocking buffer (25 mM KPO<4>, 0.2% Casein, 0.5% Boric Acid, 0.02% Sucrose, 0.1% Surfactant 10G, 0.5% PVA). After 15 min, the saturated conjugate pads were transferred to a paper towel for 1 minute. The conjugate pads were then placed on a rack to dry at 37 °C for 1 h. The blocked conjugate pads were placed in a plastic bag with desiccant and stored in a dry room. Gold-conjugated BB2 antibody DO10 was prepared by adding 10% Sucrose and 5% Trehalose to the conjugate. The gold-conjugated antibody was dispensed onto the conjugate pads by an automatic striper (Matrix 160, Kinematic Automation, Inc., Twain Harte, California, USA) at a dispensing rate of 10 pl/cm. The conjugate pads were dried at 37 °C for 1 hour, packed in a desiccated aluminum foil pouch, and stored in a dry room.

Reagent band lamination and cutting: The nitrocellulose membrane stripped with the test antibody BB7 and the control goat anti-mouse antibody was laminated to a vinyl support card (G&L Precision Die Cutting, San Jose, California, USA). A wick pad (30250, EMI Specialty Papers, Redding, Connecticut, USA) was placed on top of the support, overlapping the membrane by 2 mm. A 10 mm conjugate pad overlapped the membrane by 2 mm. A sample pad (Surewick C048 cellulose pad, Millipore, Darmstadt, Germany) was placed on top of the conjugate pad with a 15 mm overlap from the bottom of the support card. The assembled cards were cut into 4 mm strips with a cutter (CM4000, Biodot, Irvine, California, USA).

Test Swab Extraction: Swab samples were obtained from test locations with varying levels of bed bug infestation, designated as levels 0, 2, 3, 4, 5, 7, and 8, with level 0 being the lowest (i.e., no bed bugs) and 8 being the highest. Swabs were extracted in 350 µl of extraction buffer for 15 minutes at room temperature in an Eppendorf tube. Three extraction buffers were tested: Extraction Buffer 1 contained 1X Tris-HCl (pH 7.6), 0.05% NaN<3>, 0.1% BSA, and 0.1% Tween-20; Extraction buffer 2 contained 1X Tris-HCl (pH 7.6), 0.05% NaN<3>, 0.1% BSA, and 0.2% Tween-20; and extraction buffer 3 contained 1X Tris-HCl (pH 7.6), 0.05% NaN<3>, 0.25% BsA, and 0.1% Tween-20. Serial dilutions of the swab extracts were made from 1/2 to 1/4096.

Assay Procedure: 70 µl of extraction buffer (negative control) or bed bug sample in extraction buffer was pipetted onto the sample pad. The intensity of the test lines was read by eye after 15 minutes.

Extraction Buffer 1 Results: As shown in FIGS. 16-21, all bands showed positive control lines for binding of goat anti-mouse antibody to gold-conjugated BB2 antibody. All bands showed absence of test lines for the negative control band where extraction buffer was added instead of a swab sample. FIG. 16 only shows positive control lines for the level 0 swab sample dilutions since bed bug antigen was not present. In FIGS. 17-21, the positive control lines are the top lines, and the test lines showing the presence of bed bug antigen are below the positive control lines. Dirt or insoluble particles from the swabs were present near the bottom of the membranes for the Level 3, 5, 7, and 8 reagent bands. The Level 2 swab sample (FIG. 17) had test lines visible from the 1/16 dilution to the 1/2 dilution. The Level 3 swab sample (FIG. 18) had test lines visible from the 1/128 dilution to the 1/2 dilution. The Level 4 swab sample (FIG. 19) had test lines visible from the 1/1024 dilution to the 1/2 dilution. The Level 7 swab sample (FIG. 20) had test lines visible from the 1/4096 dilution to the 1/2 dilution. The Level 8 swab sample (FIG. 21) had test lines visible from the 1/2048 dilution to the 1/2 dilution. Although visually observed, some of the noted test lines are not readily apparent for certain dilutions in FIGS. 17 through 21 due to photographic limitations. All test lines were unblemished. The signal intensity of the 1/2 dilution of the level 2 swab was approximately equal to that of the 1/32 dilution of the level 3 swab, the 1/64 dilution of the level 4 swab, the 1/1024 dilution of the level 7 swab, and the 1/512 dilution of the level 8 swab. Therefore, the visible signal intensity of the 1/2 dilution of the level 2 swab was weaker, compared with that of the 1/2 dilutions of the levels 3, 4, 7, and 8 swabs. The signal of the 1/2 dilution of the level 8 swab was the strongest.

Signal intensities were also determined by using an Axxin reagent strip reader (Axxin, Fairfield, Victoria, Australia) to measure the areas of test lines for different concentrations (1/2048 to 1/2) of level 0, 2, 3, 4, 7, and 8 swab samples extracted in 350 µl of buffer 1. The results are shown in Table 2.

Table 2: Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 1)

For each level, a reading was obtained for the buffer as a negative control (i.e., the “0” concentration). The negative control (Bo) reading was divided by itself to obtain a normalized value of 1. The negative control (Bo) reading was then divided by the area of the test lines (B) for each dilution in the level, with smaller values below 1 suggesting higher amounts of bed bug antigen and values above 1 indicating no bed bug antigen. Data expressed in Bo/B are presented in Table 3.

Table 3: Bo/B Calculated from the Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 1)

Tables 2 and 3 and FIG. 22A show, in general, that the measured concentration values for the level 8 swabs (corresponding to the highest level of bed bug infestation) were the highest and the concentration values for the level 2 swabs (corresponding to the lowest level of bed bug infestation) were the lowest. Table 2 and FIG. 22B show that the level 8 swabs produced better signal intensities than the levels 2 and 4. Table 3 suggests that the level 8 swabs contained more bed bug antigen than the other levels.

Extraction Buffer 2 Results: As shown in FIGS. 23-25, all bands showed positive control lines for binding of goat anti-mouse antibody to gold-conjugated BB2 antibody. All bands showed absence of test lines for the negative control band where extraction buffer was added instead of a swab sample. In FIGS. 23-25, the positive control lines are the top lines, and the test lines showing the presence of bed bug antigen are below the positive control lines. Dirt or insoluble particles from the swabs were present near the bottom of the membranes for the level 4, 5, and 8 reactive bands. The level 4 swab sample (FIG. 23) had test lines visible from the 1/512 dilution to the 1/2 dilution. The level 5 and 8 swab samples (FIGs.

24 and 25, respectively) had test lines visible from the 1/1024 dilution to the 1/2 dilution. Although visually observed, some of the noted test lines are not readily apparent for certain dilutions in FIGS. 23 through 25 due to photographic limitations. All test lines were smeared. The signal intensity from the 1/16 dilution of the Level 4 swab was approximately equal to that from the 1/64 dilution of the Level 5 and 8 swabs. The signal intensity was weaker for the Level 4 swab and similar between the Level 5 and 8 swabs.

Signal intensities were also determined by using an Axxin reagent strip reader (Axxin, Fairfield, Victoria, Australia) to measure the areas of test lines for different concentrations (1/2048 to 1/2) of level 4, 5, and 8 swab samples extracted in 350 µl of buffer 2. The results are shown in Table 4.

Table 4: Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 2)

Data expressed as Bo/B calculated from the areas of the test lines are given in Table 5.

Table 5: Bo/B Calculated from the Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 2)

Tables 4 and 5 and FIG. 26A show that, overall, the measured values for the Level 8 swabs were the highest and the values for the Level 4 swabs were the lowest. Although the visibility of the test lines for the Level 5 swabs was similar to that for the Level 8 swabs (FIGs. 24 and 25, respectively), the Axxin reader results in Table 4 and FIG. 26A show that the Level 8 swabs produced better signal intensities than the Level 4 and 5 swabs. Table 5 suggests that the Level 8 swabs contained more bed bug antigen than the other levels.

Extraction Buffer 3 Results: As shown in FIGS. 27-31, all bands showed positive control lines for binding of goat anti-mouse antibody to gold-conjugated BB2 antibody. All bands showed absence of test lines for the negative control band where extraction buffer was added instead of a swab sample. In FIGS. 27-31, the positive control lines are the top lines, and test lines showing the presence of bed bug antigen are below the positive control lines. Dirt or insoluble particles from the swabs were present near the bottom of the membranes for the level 3, 5, 7, and 8 reactive bands. The level 2 sample (not shown) had test lines visible from the 1/8 dilution to the 1/2 dilution. The Level 3 swab sample (FIG. 27) had visible test lines from 1/128 dilution to 1/2 dilution. The Level 4 swab sample (FIG. 28) had visible test lines from 1/1024 dilution to 1/2 dilution. The Level 5 swab sample (FIG. 29) had visible test lines from 1/512 dilution to 1/2 dilution. The Level 7 swab sample (FIG.

30) had test lines visible from the 1/4096 dilution to the 1/2 dilution. The level 8 swab sample (FIG. 31) had test lines visible from the 1/2048 dilution to the 1/2 dilution. Although visually observed, some of the noted test lines are not readily apparent for certain dilutions in FIGS. 27 through 31 due to photographic limitations. All test lines were smeared. The signal intensity of the 1/2 dilution of the level 2 swab was approximately equal to that of the 1/64 dilution of the level 3 swab, the 1/64 dilution of the level 4 swab, the 1/64 dilution of the level 5 swab, the 1/1024 dilution of the level 7 swab, and the 1/512 dilution of the level 8 swab. Therefore, the visible signal intensity of the 1/2 dilution of the level 2 swab was weaker compared with that of the 1/2 dilutions of the other levels. The signal of the 1/2 dilution of the level 8 swab was the strongest.

Signal intensities were also determined by using an Axxin reagent strip reader (Axxin, Fairfield, Victoria, Australia) to measure the areas of test lines for different concentrations (1/2048 to 1/2) of level 4, 5, and 8 swab samples extracted in 350 µl of buffer 3. The results are shown in Table 6.

Table 6: Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 3)

Data expressed as Bo/B calculated from the areas of the test lines are given in Table 7.

Table 7: Bo/B Calculated from the Areas of Test Lines of Different Concentrations of Swab Samples (Buffer 3)

Tables 6 and 7 and FIG. 32A show that, in general, the measured values for level 8 swabs were the highest and those for level 2 were the lowest. Table 7 and FIG. 32A show that level 8 swabs produced better signal intensities than the other levels.

Comparison of extraction buffers: For extraction buffer 1, all test lines were clear and unstained, whereas extraction buffers 2 and 3 resulted in smeared test lines. For level 4 swabs, extraction with buffers 1 and 3 resulted in signals from dilution 1/1024, but extraction with buffer 2 resulted in signals from dilution 1/512. However, level 5 extraction with buffer 2 produced a signal at a lower concentration (dilution 1/1024) than extraction with buffer 3 (dilution 1/512). Buffers 1 and 3 gave signals at the same concentration for level 3 and 7 swabs. However, extraction of level 2 swabs with buffer 1 resulted in a lower concentration signal (1/16 dilution) than extraction with buffer 3 (1/8 dilution). Overall, the measured values for all test level swabs were higher for extraction with buffer 1 than for buffers 2 and 3.

Axxin Reader Precision Study: Nine replicates of Extraction Buffer 1 as a negative control and nine replicates each of 1/2048, 1/512, and 1/128 dilutions of Level 7 swabs extracted with Buffer 1 were prepared and the test line areas were measured using an Axxin Reagent Strip Reader (Axxin, Fairfield, Victoria, Australia). The test line areas are shown in Table 8 and FIG.

34.

Table 8: Accuracy Study Results of Replicated Test Areas

The percentage coefficient of variation (% CV) was less than 20%, which is a good % CV for Axxin measurements.

Conclusion

Claims (13)

1. An electronic detection device for analyzing a test fluid to determine past or present bed bug infestations, comprising a lateral flow assay processed within the detection device, wherein the lateral flow assay comprises a reagent strip for receiving the test fluid near a first end of the reagent strip, wherein the reagent strip comprises: (a) a reagent portion comprising monoclonal antibodies or antigen-binding fragments thereof that are conjugated to colored particles and are capable of binding to bed bug antigens within the test fluid to form bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7], and (b) a reaction portion comprising immobilized monoclonal antibodies or antigen-binding fragments thereof that are capable of binding to bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7]. 2. A reagent strip for detecting past or present infestations of bed bugs comprising, in order, a first end, a reagent portion containing colored particles, a reaction portion, and a second end, wherein the reagent strip is configured to receive a test fluid proximate the first end and: (a) the reagent portion comprises monoclonal antibodies or antigen-binding fragments thereof that are conjugated to the colored particles and are capable of binding to bed bug antigens within the test fluid to form bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession Number pTa-122644 [BB2] or Accession Number PTA-122645 [BB7], and (b) the reaction portion comprises immobilized monoclonal antibodies or antigen-binding fragments thereof that are capable of binding to bed bug molecules, wherein the monoclonal antibodies or antigen-binding fragments thereof comprise heavy and light chain complementarity determining regions (CDRs) of an antibody produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7]. 3. The electronic detection device of claim 1 or the reagent strip of claim 2, wherein the reagent strip comprises: (a) a backing strip comprising a wick for receiving and absorbing the test fluid, (b) a porous carrier strip comprising a reagent pad affixed to the wick, wherein the reagent pad comprises the reagent portion, (c) a line-forming zone comprising the reaction portion, and (d) an absorption pad at the opposite end portion of the reagent strip from the wick. 4. The electronic detection device of claim 1 or 3, or the reagent band of claim 2 or 3, wherein the monoclonal antibodies or antigen-binding fragments thereof in the reagent portion and/or the reaction portion comprise heavy and light chain variable regions of the antibody produced by a hybridoma deposited with the ATCC under Accession Number<p>T<a>-122644 [BB2] or Accession Number PTA-122645 [BB7]. 5. The electronic detection device of claim 1 or 3, or the reagent band of claim 2 or 3, wherein the monoclonal antibodies or antigen-binding fragments thereof in the reagent portion and/or the reaction portion comprise heavy and light chains of the antibody produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2] or Accession Number PTA-122645 [BB7]. 6. The electronic detection device of any one of claims 1 or 3 to 5, or the reagent band of any one of claims 2 to 5, wherein the monoclonal antibodies or antigen-binding fragments thereof in the reagent portion and/or the reaction portion are capable of binding to a bed bug antigen in a whole bed bug lysate or a collection paper extract comprising bed bug litter material. 7. The electronic detection device of any one of claims 1 or 3 to 6, or the reagent band of any one of claims 2 to 6, wherein the reagent portion comprises monoclonal antibodies produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], or an antigen-binding fragment thereof. 8. The electronic detection device of any one of claims 1 or 3 to 6, or the reagent band of any one of claims 2 to 6, wherein the reagent portion comprises monoclonal antibodies produced by a hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], or an antigen-binding fragment thereof. 9. The electronic detection device of any one of claims 1 or 3 to 8, or the reagent band of any one of claims 2 to 8, wherein the reaction portion comprises monoclonal antibodies produced by a hybridoma deposited with the ATCC under Accession Number PTA-122644 [BB2], or an antigen-binding fragment thereof. 10. The electronic detection device of any one of claims 1 or 3 to 8, or the reagent band of any one of claims 2 to 8, wherein the reaction portion comprises monoclonal antibodies produced by a hybridoma deposited with the ATCC under Accession Number PTA-122645 [BB7], or an antigen-binding fragment thereof. 11. The electronic detection device of any one of claims 1 or 3 to 10, or the reagent strip of any one of claims 2 to 10, wherein the colored particles in the reagent portion are colloidal gold, a latex microsphere or a fluorescent label. 12. A method of analyzing a test fluid to determine past or present bed bug infestations, comprising contacting the test fluid with the reagent strip of the electronic detection device of any one of claims 1 or 3 to 11 or the reagent strip of any one of claims 2 to 11. 13. A kit comprising the electronic detection device of any one of claims 1 or 3 to 11 or the reagent strip of any one of claims 2 to 11.